The present invention is directed to methods for the identification of new anti-cancer agents that inhibit an epithelial-mesenchymal transition (EMT) in tumor cells, and their use in treating cancer patients, particularly in combination with other agents such as EGFR or IGF-1R kinase inhibitors that can be less effective at inhibiting tumor cells that have undergone an EMT. Cancer is a generic name for a wide range of cellular malignancies characterized by unregulated growth, lack of differentiation, and the ability to invade local tissues and metastasize. These neoplastic malignancies affect, with various degrees of prevalence, every tissue and organ in the body.
An anti-neoplastic drug would ideally kill cancer cells selectively, with a wide therapeutic index relative to its toxicity towards non-malignant cells. It would also retain its efficacy against malignant cells, even after prolonged exposure to the drug. Unfortunately, none of the current chemotherapies possess such an ideal profile. Instead, most possess very narrow therapeutic indexes. Furthermore, cancerous cells exposed to slightly sub-lethal concentrations of a chemotherapeutic agent will very often develop resistance to such an agent, and quite often cross-resistance to several other antineoplastic agents as well.
A multitude of therapeutic agents have been developed over the past few decades for the treatment of various types of cancer. The most commonly used types of anticancer agents include: DNA-alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate, a folate antagonist, and 5-fluorouracil, a pyrimidine antagonist), microtubule disrupters (e.g., vincristine, vinblastine, paclitaxel), DNA intercalators (e.g., doxorubicin, daunomycin, cisplatin), and hormone therapy (e.g., tamoxifen, flutamide). More recently, gene targeted therapies, such as protein-tyrosine kinase inhibitors have increasingly been used in cancer therapy (de Bono J. S. and Rowinsky, E. K. (2002) Trends in Mol. Medicine. 8:S19-S26; Dancey, J. and Sausville, EBA. (2003) Nature Rev. Drug Discovery 2:92-313). Such approaches, such as the EGFR kinase inhibitor erlotinib, are generally associated with reduced toxicity compared with conventional cytotoxic agents. They are therefore particularly appropriate for use in combination regimens. In pancreatic cancer, phase III trials have shown that first-line erlotinib treatment in combination with gemcitabine improves survival.
The epidermal growth factor receptor (EGFR) family comprises four closely related receptors (HER1/EGFR, HER2, HER3 and HER4) involved in cellular responses such as differentiation and proliferation. Over-expression of the EGFR kinase, or its ligand TGF-alpha, is frequently associated with many cancers, including breast, lung, colorectal, ovarian, renal cell, bladder, head and neck cancers, glioblastomas, and astrocytomas, and is believed to contribute to the malignant growth of these tumors. A specific deletion-mutation in the EGFR gene (EGFRvIII) has also been found to increase cellular tumorigenicity. Activation of EGFR stimulated signaling pathways promote multiple processes that are potentially cancer-promoting, e.g. proliferation, angiogenesis, cell motility and invasion, decreased apoptosis and induction of drug resistance. Increased HER1/EGFR expression is frequently linked to advanced disease, metastases and poor prognosis. For example, in NSCLC and gastric cancer, increased HER1/EGFR expression has been shown to correlate with a high metastatic rate, poor tumor differentiation and increased tumor proliferation.
Mutations which activate the receptor's intrinsic protein tyrosine kinase activity and/or increase downstream signaling have been observed in NSCLC and glioblastoma. However the role of mutations as a principle mechanism in conferring sensitivity to EGF receptor inhibitors, for example erlotinib (TARCEVA®) or gefitinib (IRESSA™), has been controversial. Recently, a mutant form of the full length EGF receptor has been reported to predict responsiveness to the EGF receptor tyrosine kinase inhibitor gefitinib (Paez, J. G. et al. (2004) Science 304:1497-1500; Lynch, T. J. et al. (2004) N. Engl. J. Med. 350:2129-2139). Cell culture studies have shown that cell lines which express the mutant form of the EGF receptor (i.e. H3255) were more sensitive to growth inhibition by the EGF receptor tyrosine kinase inhibitor gefitinib, and that much higher concentrations of gefitinib was required to inhibit the tumor cell lines expressing wild type EGF receptor. These observations suggests that specific mutant forms of the EGF receptor may reflect a greater sensitivity to EGF receptor inhibitors, but do not identify a completely non-responsive phenotype.
Erlotinib (e.g. erlotinib HCl, also known as TARCEVA® or OSI-774) is an orally available inhibitor of EGFR kinase. In vitro, erlotinib has demonstrated substantial inhibitory activity against EGFR kinase in many human tumor cell lines. In a phase III trial, erlotinib monotherapy significantly prolonged survival, delayed disease progression and delayed worsening of lung cancer-related symptoms in patients with advanced, treatment-refractory NSCLC (Shepherd, F. et al. (2005) N. Engl. J. Med. 353(2):123-132). In November 2004 the U.S. Food and Drug Administration (FDA) approved TARCEVA® for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) after failure of at least one prior chemotherapy regimen.
The development for use as anti-tumor agents of compounds that directly inhibit the kinase activity of IGF-1R, as well as antibodies that reduce IGF-1R kinase activity by blocking IGF-1R activation or antisense oligonucleotides that block IGF-1R expression, are also areas of intense research effort (e.g. see Larsson, O. et al (2005) Brit. J. Cancer 92:2097-2101; Ibrahim, Y. H. and Yee, D. (2005) Clin. Cancer Res. 11:944s-950s; Mitsiades, C. S. et al. (2004) Cancer Cell 5:221-230; Camirand, A. et al. (2005) Breast Cancer Research 7:R570-R579 (DOI 10.1186/bcr1028); Camirand, A. and Pollak, M. (2004) Brit. J. Cancer 90:1825-1829; Garcia-Echeverria, C. et al. (2004) Cancer Cell 5:231-239).
IGF-1R is a transmembrane RTK that binds primarily to IGF-1 but also to IGF-II and insulin with lower affinity. Binding of IGF-1 to its receptor results in receptor oligomerization, activation of tyrosine kinase, intermolecular receptor autophosphorylation and phosphorylation of cellular substrates (major substrates are IRS1 and Shc). The ligand-activated IGF-1R induces mitogenic activity in normal cells and plays an important role in abnormal growth. A major physiological role of the IGF-1 system is the promotion of normal growth and regeneration. Overexpressed IGF-1R (type 1 insulin-like growth factor receptor) can initiate mitogenesis and promote ligand-dependent neoplastic transformation. Furthermore, IGF-1R plays an important role in the establishment and maintenance of the malignant phenotype. Unlike the epidermal growth factor (EGF) receptor, no mutant oncogenic forms of the IGF-1R have been identified. However, several oncogenes have been demonstrated to affect IGF-1 and IGF-1R expression. The correlation between a reduction of IGF-1R expression and resistance to transformation has been seen. Exposure of cells to the mRNA antisense to IGF-1R RNA prevents soft agar growth of several human tumor cell lines. IGF-1R abrogates progression into apoptosis, both in vivo and in vitro. It has also been shown that a decrease in the level of IGF-1R below wild-type levels causes apoptosis of tumor cells in vivo. The ability of IGF-1R disruption to cause apoptosis appears to be diminished in normal, non-tumorigenic cells.
The IGF-1 pathway in human tumor development has an important role. IGF-1R overexpression is frequently found in various tumors (breast, colon, lung, sarcoma) and is often associated with an aggressive phenotype. High circulating IGF1 concentrations are strongly correlated with prostate, lung and breast cancer risk. Furthermore, IGF-1R is required for establishment and maintenance of the transformed phenotype in vitro and in vivo (Baserga R. Exp. Cell. Res., 1999, 253, 1-6). The kinase activity of IGF-1R is essential for the transforming activity of several oncogenes: EGFR, PDGFR, SV40 T antigen, activated Ras, Raf, and v-Src. The expression of IGF-1R in normal fibroblasts induces neoplastic phenotypes, which can then form tumors in vivo. IGF-1R expression plays an important role in anchorage-independent growth. IGF-1R has also been shown to protect cells from chemotherapy-, radiation-, and cytokine-induced apoptosis. Conversely, inhibition of endogenous IGF-1R by dominant negative IGF-1R, triple helix formation or antisense expression vector has been shown to repress transforming activity in vitro and tumor growth in animal models.
During most cancer metastases, an important change occurs in a tumor cell known as the epithelial-mesenchymal transition (EMT) (Thiery, J. P. (2002) Nat. Rev. Cancer 2:442-454; Savagner, P. (2001) Bioessays 23:912-923; Kang Y. and Massague, J. (2004) Cell 118:277-279; Julien-Grille, S., et al. Cancer Research 63:2172-2178; Bates, R. C. et al. (2003) Current Biology 13:1721-1727; Lu Z., et al. (2003) Cancer Cell. 4(6):499-515)). EMT does not occur in healthy cells except during embryogenesis. Epithelial cells, which are bound together tightly and exhibit polarity, give rise to mesenchymal cells, which are held together more loosely, exhibit a loss of polarity, and have the ability to travel. These mesenchymal cells can spread into tissues surrounding the original tumor, as well as separate from the tumor, invade blood and lymph vessels, and travel to new locations where they divide and form additional tumors. Recent research has demonstrated that epithelial cells respond well to EGFR and IGF-1R kinase inhibitors, but that after an EMT the resulting mesenchymal-like cells are much less sensitive to such inhibitors. (e.g. Thompson, S. et al. (2005) Cancer Res. 65(20):9455-9462; U.S. Patent Application 60/997,514). Thus there is a pressing need for anti-cancer agents that can prevent or reverse tumor cell EMT events (e.g. stimulate a mesenchymal to epithelial transition (MET)), or inhibit the growth of the mesenchymal-like tumor cells resulting from EMT. Such agents should be particularly useful when used in conjunction with other anti-cancer drugs such as EGFR and IGF-1R kinase inhibitors.
Snail is a Zn-finger transcriptional repressor and a master regulator of the epithelial-mesenchymal transition (EMT) in development and cancer progression [Peinado, H., D. et al. Nat Rev Cancer, 2007. 7(6): p. 415-28]. Snail has been described as a direct repressor of E-cadherin expression through binding to the conserved E-boxes in the proximal promoter region. Snail can also upregulate the expression of ZEB1, another EMT driver and E-box binding repressor of E-cadherin and other epithelial genes [Aigner, K., et al., Oncogene, 2007, 26(49):6979-88; Guaita, S., et al., J Biol Chem, 2002. 277(42): p. 39209-16]. The expression of Snail induces complete EMT and increases cellular migration and invasion in different physiological and pathological settings. Recent studies have implicated Snail expression and function with tumor recurrence of breast cancer [Moody, S. E., et al. Cancer Cell, 2005. 8(3): p. 197-209] in addition to increased metastasis and chemoresistance of pancreatic cancer [Yin, T., et al., J Surg Res, 2007. 141(2): p. 196-203].
Stimuli that induce EMT such as TGFβ, HGF, Shh, Wnt seem to do so by upregulating the expression [Cho, H. J., et al., Biochem Biophys Res Commun, 2007. 353(2): p. 337-43; Grotegut, S., et al., Embo J, 2006. 25(15): p. 3534-45; Li, X., et al., Oncogene, 2006. 25(4): p. 609-21] and stability [Yook, J. I., et al., Nat Cell Biol, 2006. 8(12): p. 1398-406] of Snail. GSK-3β phosphorylates Snail at two consensus motifs; phosphorylation of the first motif (residues 96-104) regulates Snail's ubiquitination and degradation while phosphorylation of the second motif (residues107-119) favors its exclusion from the nucleus [Zhou, B. P., et al., Nat Cell Biol, 2004. 6(10): p. 931-40]. On the other hand, it has been demonstrated that phosphorylation of Snail by the p21-activated kinase 1 (PAK1) on Ser-246 favors shuttling of Snail to the nucleus thereby enhancing its repressor function [Yang, Z., et al., Cancer Res, 2005. 65(8): p. 3179-84].
PAK1 is a group I p21-activated serine/threonine kinase and is highly homologous (˜92% amino acid identity in the kinase domain) to the two other members PAK2 and PAK3. While PAK1 and PAK2 show universal expression, PAK3's expression is restricted to central nervous system tissues. PAK1-3 are generally thought to phosphorylate overlapping sets of substrates. In addition to Snail, two other EMT-related proteins have recently been identified as putative PAK1 substrates, integrin-linked kinase ILK and heterogeneous nuclear ribonucleoprotein hnRNP-A/B (a.k.a. CBF-A) [Acconcia, F., et al., Proc Natl Acad Sci USA, 2007. 104(16): p. 6782-7; Meng, Q., et al. Proc Natl Acad Sci USA, 2007. 104(14): p. 5866-71]. Both ILK and hnRNP-A/B cause full EMT upon overexpression in epithelial cells [Somasiri, A., et al., J Cell Sci, 2001. 114(Pt 6): p. 1125-36; Venkov, C. D., et al., J Clin Invest, 2007. 117(2): p. 482-91]. It has not been shown whether any of these substrates is phosphorylatable by PAK2 and hence whether PAK2 activity can impact EMT. PAK2 has however been previously linked to the TGFβ-mediated morphological transformation of fibroblasts into myofibroblasts [Wilkes, M. C., et al. Mol Cell Biol, 2003. 23(23): p. 8878-89], a process akin to EMT in epithelial cells. The mentioned study demonstrated that TGFβ activates PAK2 only in fibroblast cells and not in epithelial cells [Wilkes, M. C., et al., Mol Cell Biol, 2003. 23(23): p. 8878-89]. PAK2 kinase inhibition has also been suggested as a potential method for treating cancer (US Patent Application Publication US 2005/0080002).
The invention described herein provides methods for the identification of new anti-cancer agents that inhibit PAK2 and EMT in tumor cells, and their use in treating cancer patients. The invention described herein also provides new anti-cancer combination therapies that are an improvement on the efficacy of either EGFR kinase inhibitors or or IGF-1R kinase inhibitors when administered alone. In particular, the present invention is directed to methods of combined treatment of cancer with an epidermal growth factor receptor (EGFR) kinase inhibitor (or an IGF-1R kinase inhibitor) and an inhibitor of PAK2 kinase.